Platelets are a component of blood comprised of anucleate megakaryocyte fragments that circulate in the blood for about 10 days (van der Meer, Pietersz et al. 2001; Dijkstra-Tiekstra 2004). As they age in the circulatory system platelets are known to undergo biochemical changes that eventually leads to their clearance in the spleen and liver. When separated as a component of whole blood, platelets are routinely concentrated, resuspended in plasma and/or platelet additive solutions, leukoreduced by passage through a filtration device and stored in platelet storage bags which are kept on flatbed agitators for 5 to 7 days at a temperature of 22° C.
The measurement of pH and other parameters during preparation and storage of blood components are necessary in order to provide a safe and effective product. For example the storage of platelets at 22° C. requires testing for the presence of microbiological contamination to prevent undesired side effects such as sepsis as a result of infusion into the patient. Under the American Association of Blood Banks (A.A.B.B.) standard 5.1.5.1, blood banks or transfusion services are instructed to have methods to limit and detect bacterial contamination in all platelets. The growth of bacteria in platelet concentrates (PCs) can be monitored by utilizing reagent dipsticks for pH and glucose however the time of detection following inoculation with a low dose of organisms (e.g. 50 colony forming units/mL) varies from organism to organism, limiting this method's sensitivity and specificity given the shelf life of the product (Brecher, Hogan et al. 1994). Even in light of these limitations many centers have moved to time of issue tests utilizing the measurement of pH and glucose with a handheld device, pH paper, or in combination on a multi-reagent dipstick as surrogate markers for bacterial contamination (Burstain, Brecher et al. 1997; Yazer and Triulzi 2005).
In one method of non-invasive bacterial detection, changes in pH or the production of CO2 was detected in clinical specimens by culturing the specimens with a sterile liquid growth medium in a transparent sealed container (Calandra et al., U.S. Pat. No. 5,094,955). The main disadvantage of this method is the requirement for sampling of a clinical specimen and introduction of the sample into a separate culture vessel at one point in time. The sampling is disadvantageous because the contaminating organisms may not be included in the sample volume, resulting in a false negative test. A similar method employed a sensor to detect microbial organisms growing in a liquid environment with the microbial colonies immediately available for further testing by virtue of the design of the culture vessel (Jeffrey et al., U.S. Pat. No. 5,976,827). This general method by which culture-based bacterial detection systems function is currently used for detection of microorganisms in PCs (Brecher, Means et al. 2001; McDonald, Pearce et al. 2005). A device for measuring the pH of PCs non-invasively by a fluorescence-based interrogation of the bag contents has been described in the WO 2006/023725 (PCT/US2005/029559), expressly incorporated herein by reference in its entirety.
Additional important reasons to measure pH in the quality control of PCs include its correlation with in vivo viability following transfusion into patients (Moroff, Friedman et al. 1982; Solberg, Holme et al. 1986; Holme 1998; Rinder, Snyder et al. 2003). pH Values below 6.2 in PCs have been correlated with poor in vivo recovery in transfusion studies (Murphy and Gardner 1971; Slichter and Harker 1976; Murphy 1985), while loss of recovery in vivo at pH values above 7.2 has been shown (Murphy and Gardner 1975). The platelet yield in PCs is also an important quality control parameter because it establishes the therapeutic dosage of the product and may influence the levels of metabolic activity measured within the storage bags. There have been several studies showing maintenance of pH values indicative of good platelet function up to 7 or 8 days in PCs stored in mixtures of additive solutions and plasma (Klinger 1996), provided that the platelet content was <4×1011(de Wildt-Eggen, Schrijver et al. 1998). These latter studies showed that a more rapid decline in pH in a PC may correlate with higher platelet concentrations. A similar correlation of a more rapid decline in pH over the five day storage period was inferred in apheresis derived PCs obtained from machines that consistently produce product with higher platelet counts (Tudisco, Jett et al. 2005).
The storage of platelets in BTHC (n-butyryl, tri-n-hexyl citrate) PVC containers presents a number of advantages with respect to platelet health. These containers have several desirable characteristics for this medical application including low toxicity and permeability to water, O2, and CO2 in the desired ranges. The BTHC plasticized PVC material also has high permeability for CO2, excess of which in some cases leads to difficulties storing PCs which have high platelet counts. Depending on storage conditions the pH of PCs can change rapidly due to off-gassing of dissolved CO2. The Council of Europe guidelines for platelet storage conditions require the pH to be in the range of 6.4-7.4 at 37° C. (6.6-7.6 at 22° C.).
Optical sensors (optrodes) for measuring pH are well known. Certain aromatic organic compounds (like phenolphthalein) change color with pH and can be immobilized on solid supports to form “pH paper.” These visual indicators are easy to use, but do not provide a quantitative reading. The color changes can be difficult to distinguish accurately, and can be masked by colored analyte. Fluorescent indicators have also been used as optical sensors. pH Sensitive fluorescent dyes can be immobilized on solid supports and generally are more sensitive in comparison to the simple colorimetric (absorbance or reflectance based) indicators. The improved sensitivity of fluorescent indicators allows the solid support to be miniaturized, and this has been used to advantage in development of fiber optic sensor devices for measuring pH, CO2, and O2 parameters in blood.
A specific need in the medical industry exists for accurate pH measurement of blood and blood products. The pH of blood or other bodily fluids (pleural effusions) can be associated with certain physiologic responses associated with pathology. Blood gas analyzers are common critical care instruments. Depending on storage conditions, the pH of separated blood components (plasma, platelets) can change rapidly due to off-gassing of dissolved CO2 from the enriched venous blood that is collected from a donor. Platelets in particular are metabolically active, and generate lactic acid during storage at 20° C. to 22° C. European quality guidelines for platelets prepared by the “buffycoat method” require pH of stored platelets to be pH 6.8-7.4 at 37° C. (7.0-7.6 at 22° C.).
Seminaphthofluorescein (SNAFL) compounds and the related seminaphthorhodafluor (SNARF) compounds are commercially available ratiometric fluors (Molecular Probes, Inc., Eugene, Oreg.; see, for example, U.S. Pat. No. 4,945,171) and their synthesis and spectral properties have been described. These compounds have advantages including long wavelength absorbance that can be efficiently excited with LED light sources. Relevant acid/base equilibria and associated spectral properties are shown below.

Deprotonation of the naphthol structure of SNAFL dyes gives a naphtholate molecule with longer wavelength fluorescence emission. The pKa is the pH value where the two molecular species form in equal amounts. SNAFL compounds with reactive linker groups that allow their conjugation to other molecules of interest are also commercially available.
Various methods have been used to immobilize “ratiometric” dyes to solid supports for use in fiber optic pH detectors. Carboxynaphthofluorescein (CNF) has been conjugated to aminoethyl-cellulose and this material was glued to polyester (Mylar) films to make sensing membranes for optrodes. The pKa of this material was determined to be 7.41, slightly lower than the free CNF (pKa 7.62). The use of tetraethoxysilane to trap CNF in a sol-gel glass that was formed on glass cover slips has also been reported. The pKa of this material was determined to be 7.46. A 9-chloro substituted SNAFL analog (SNAFL-2) has been reacted with polyvinylamine and the residual amino groups crosslinked with a photocrosslinker to form a gel-like coating on acrylic fibers. The pKa of this fiber-optic sensor was determined to be 7.14, significantly lower than the published pKa of the free SNAFL compound (pKa ˜7.7). This shows that molecular environment and linker structure surrounding the immobilized dye can alter the performance of a pH detector.
Despite the advances made in the detection of pH noted above, there exists a need for improved methods and devices for monitoring the chemical environment in a sealed sterile container, continually or at discrete time intervals, in order to better understand the types and levels of metabolic activities within the container, as well as their origin. The present invention seeks to fulfill this need and provides further related advantages.